Neural stem cell temporal patterning and brain tumour growth rely on oxidative phosphorylation
- The Gurdon Institute, United Kingdom
- University of Cambridge, United Kingdom
Figures
Figure 1 with 2 supplements
Brain tumours require OxPhos for growth.
(a–g) phospho Histone H3 (pH3) staining in the CNS of third instar larvae (L3) with NSC-specific expression (Wor-GAL4;Tub-GAL80ts) of control RNAi (a), Pros-RNAi (b,c), aPKC-CAAX (d,e) or Brat-RNAi (…
Figure 1—figure supplement 1
Brain tumours require OxPhos and glycolysis for their growth.
(a,b) pH3 in the CNS of L3 larvae. Maximum intensity projections through the CNS; dashed lines outline the CNS. (c) Brain size (area of CNS, maximum intensity projections) from L3 larvae. Datapoints …
Figure 1—figure supplement 2
OxPhos RNAi in NSCs affects mitochondrial function.
(a–c) ATPsynα (mitochondria, Complex V subunit) and GFP (mitochondria in NSCs and their progeny; Wor-GAL4 >Mito GFP) staining in the VNC of L3 larvae with NSC-specific expression of the indicated …
Figure 2 with 4 supplements
OxPhos inhibition decreases NSC proliferation.
(a–c) pH3 staining in the CNS of L3 larvae. Maximum intensity projections through the entire CNS; dashed lines outline the CNS. (d) Brain size from L3 larvae. (e,f) Mitotic index (e) and 15 min EdU …
Figure 2—figure supplement 1
OxPhos inhibition does not affect body size.
Wandering larvae (a,b) or pupae (c,d) with NSC-specific expression of the indicated RNAi. Length is shown in millimetres (mm). Datapoints indicate individual organisms from one biological replicate. …
Figure 2—figure supplement 2
OxPhos inhibition does not increase apoptosis.
(a–d) pH3 staining in the CNS of L3 larvae with NSC-specific expression of the indicated RNAi. Maximum intensity projections through the entire CNS; dashed lines outline the CNS. (e–j) TUNEL, GFP …
Figure 2—video 1
Mitochondrial dysfunction increases cell cycle length.
Time-lapse imaging of NSCs in the early third instar larval VNC (48 hr ALH at 29°C) expressing either two copies of UAS-mCD8-GFP (Control, left), or one copy of UAS-mCD8-GFP and UAS-ND75-TRIP …
Figure 2—video 2
Mitochondrial dysfunction increases cell cycle length.
Time-lapse imaging of NSCs in the late third instar larval VNC (wandering, 72 hr ALH at 29°C) expressing either one copy of UAS-mCD8-GFP (Control, left), or one copy of UAS-mCD8-GFP and …
Figure 3 with 2 supplements
OxPhos is required for temporal patterning of NSC and their progeny.
(a) Scheme of the major temporal transitions in larval NSCs. (b–d) Dpn and Imp expression in the VNC of L3 larvae. Arrowheads indicate Imp-positive NSCs. (e) Percentage of Dpn-positive NSCs in the …
Figure 3—figure supplement 1
Mitochondrial dysfunction in NSCs delays temporal patterning.
(a–c) Overview of Imp and Syp in the thoracic VNC of L3 larvae. Single confocal sections at comparable depths through the ventral side of the VNC; dashed lines outline the VNC; arrowheads indicate …
Figure 3—figure supplement 2
Delayed temporal patterning of NSCs affects their progeny.
(a–c) Overview of Chinmo and Broad in the thoracic VNC of L3 larvae. Single confocal sections at comparable depths through the ventral side of the VNC; dashed lines outline the VNC. (d–f) RFP …
Figure 4 with 2 supplements
G1/S progression drives temporal patterning.
(a–f) Dpn (NSCs), pH3 (mitosis) and Imp in the VNC of L3 larvae after NSC-specific expression of the indicated transgene. Arrowheads indicate Imp-positive NSCs. (g) Scheme depicting activity of the …
Figure 4—figure supplement 1
G1/S and G2/M delay results in smaller brains.
(a) size (area of CNS maximum intensity projections) of pharate adult CB. (b,c) Immunostaining for Dpn (NSCs), Chinmo and Broad in the VNC of wildtype L3 larvae or larvae with NSC-specific …
Figure 4—figure supplement 2
AMPK deletion does not rescue the temporal patterning defect caused by OxPhos inhibition.
(a,b) Dpn (NSCs), RFP (negatively marked AMPK-/- clones) and Imp in the VNC of L3 larvae after heatshock at 0hALH. Arrowheads indicate Dpn-positive NSCs. Dashed outlines mark RFP-negative clones. …
Figure 5 with 1 supplement
NSCs require OxPhos for termination of proliferation.
(a–g) ElaV (neurons), GFP (NSCs, Wor-GAL4 >mCD8 GFP), Dpn (NSCs) and pH3 (mitosis) in the pharate adult CB or VNC. Maximum intensity projections through the CB or VNC; dashed lines mark the outline …
Figure 5—figure supplement 1
Adult neurogenesis upon OxPhos knockdown and G1/S delay.
(a–d) ElaV (neurons), GFP (NSCs, Wor-GAL4 >mCD8 GFP), Dpn (NSCs) and pH3 (mitosis) in the pharate adult CB (a–c) or VNC (d). Maximum intensity projections through the CB or VNC; dashed lines outline …
Figure 6
Model of the role of OxPhos in Drosophila NSCs and tumour cells.
We propose a model, whereby highly proliferative Drosophila NSCs also rely on OxPhos for most aspects of their behaviour. In particular, the G1/S transition depends on OxPhos activity and …
Author response image 1
Author response image 2
Author response image 3
Tables
Key resources table
| Reagent type (species) | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (D. melanogaster) | mCherry-TRIP | BDSC | RRID: BDSC_35785 | Control RNAi |
| Genetic reagent (D. melanogaster) | w1118;+;+ | BDSC | RRID: BDSC_3605 | |
| Genetic reagent (D. melanogaster) | ND75-TRIP | BDSC | RRID: BDSC_33911 | Complex I RNAi |
| Genetic reagent (D. melanogaster) | Blw-TRIP | BDSC | RRID: BDSC_28059 | Complex V RNAi |
| Genetic reagent (D. melanogaster) | Pros-TRIP | BDSC | RRID: BDSC_42538 | |
| Genetic reagent (D. melanogaster) | Brat-TRIP | BDSC | RRID: BDSC_28590 | |
| Genetic reagent (D. melanogaster) | ND42-TRIP | BDSC | RRID: BDSC_32998 | |
| Genetic reagent (D. melanogaster) | ND51-TRIP | BDSC | RRID: BDSC_36701 | |
| Genetic reagent (D. melanogaster) | Blw-RNAi-KK | VDRC | 34663 | |
| Genetic reagent (D. melanogaster) | ATPsynβ-TRIP | BDSC | RRID: BDSC_28056 | |
| Genetic reagent (D. melanogaster) | ATPsynγ-TRIP | BDSC | RRID: BDSC_28723 | |
| Genetic reagent (D. melanogaster) | ATPsynO-TRIP | BDSC | RRID: BDSC_43265 | |
| Genetic reagent (D. melanogaster) | PFK-TRIP | BDSC | RRID: BDSC_34336 | |
| Genetic reagent (D. melanogaster) | Aldolase-TRIP | BDSC | RRID: BDSC_26301 | |
| Genetic reagent (D. melanogaster) | PyK-TRIP | BDSC | RRID: BDSC_35218 | |
| Genetic reagent (D. melanogaster) | PGK-RNAi-KK | VDRC | 110081 | |
| Genetic reagent (D. melanogaster) | UASp-EGFP-Myt1 | BDSC | RRID: BDSC_65393 | |
| Genetic reagent (D. melanogaster) | UASt-dWee1 | (Price et al., 2002) PMID: 12072468 | ||
| Genetic reagent (D. melanogaster) | UASt-Dap | (Lane et al., 1996) PMID: 8980229 | ||
| Genetic reagent (D. melanogaster) | UAS-Rbf-280 | (Duman-Scheel et al., 2002) PMID: 12015606 | ||
| Genetic reagent (D. melanogaster) | UASt-aPKC.CAAXWT | (Lee et al., 2006; Sotillos et al., 2004) PMID: 16357871, 15302858 | ||
| Genetic reagent (D. melanogaster) | UAS-mito-HA-GFP,e1 | BDSC | RRID: BDSC_8443 | |
| Genetic reagent (D. melanogaster) | UAS-AT1.03-NL on III | (Tsuyama et al., 2013) PMID: 23875533 | ||
| Genetic reagent (D. melanogaster) | UAS-AT1.03-RK on III | (Tsuyama et al., 2013) PMID: 23875533 | ||
| Genetic reagent (D. melanogaster) | UAS-GFP-E2F1.1–230, UAS-mRFP1-NLS-CycB.1–266 | (Zielke et al., 2014) PMID: 24726363 | Fly FUCCI | |
| Genetic reagent (D. melanogaster) | Worniu-GAL4 on II | (Albertson et al., 2004) PMID: 15536119 | ||
| Genetic reagent (D. melanogaster) | Cas::GFP FlyFos line | VDRC | 318476 | |
| Genetic reagent (D. melanogaster) | Ubi-FRT-Stop-FRT-GFP | BDSC | RRID: BDSC_32251 | |
| Genetic reagent (D. melanogaster) | Imp8 | (Munro et al., 2006) PMID: 16476777 | Imp mutant | |
| Genetic reagent (D. melanogaster) | Ampkα3 | (Haack et al., 2013) PMID: 24337115 | AMPK mutant | |
| Antibody | rat anti-PH3 (monoclonal) | Abcam | ab10543 RRID: AB_2295065 | IF, 1/500 |
| Antibody | rabbit anti-PH3 (polyclonal) | Merck Millipore | 06–570 RRID: AB_310177 | IF, 1/500 |
| Antibody | guinea pig anti-Dpn (polyclonal) | James Skeath | IF, 1/10,000 | |
| Antibody | rabbit anti-Imp (polyclonal) | (Geng and Macdonald, 2006) PMID: 17030623 | IF, 1/600 | |
| Antibody | guinea pig anti-Syp (polyclonal) | (McDermott et al., 2012) PMID: 23213441 | IF, 1/1000 | |
| Antibody | chicken anti-GFP (polyclonal) | Abcam | ab13970 RRID: AB_300798 | IF, 1/2000 |
| Antibody | rat anti-Mira (polyclonal) | Chris Doe | IF, 1/500 | |
| Antibody | rat anti-Chinmo (polyclonal) | (Wu et al., 2012) PMID: 22814608 | IF, 1/500 | |
| Antibody | mouse anti-Broad (monoclonal) | DSHB | 25E9.07 | IF, 1/100 |
| Antibody | rabbit anti-RFP (polyclonal) | Abcam | ab62341 RRID: AB_945213 | IF, 1/500 |
| Antibody | rat anti-ElaV (monoclonal) | DSHB | 7E8A10 | IF, 1/100 |
| Antibody | mouse anti-Sevenup (polyclonal) | (Kanai et al., 2005) PMID: 15691762 | IF, 1/200 | |
| Antibody | mouse anti-ATPsynα (monoclonal) | Abcam | ab14748 RRID: AB_301447 | IF, 1/100 |
| Antibody | GFP-booster Atto647N | Chromotek | gba647n RRID: AB_2629215 | IF, 1/500 for STED |
| Commercial assay or kit | ApopTag Red In Situ Apoptosis Detection kit | Merkc Millipore | S7165 | |
| Commercial assay or kit | Click-iT EdU Alexa Fluor 647 Imaging Kit | Invitrogen | C10340 | |
| Chemical compound, drug | 2-deoxyglucose | Sigma | D8375 | 200 mM final concentration |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.47887.019
